The production of covalently closed circular DNA in pure form has become increasingly important in the past two decades. The development of DNA vaccines in the last few years has added to this demand. The purification protocol for ccc DNA requires its separation from the genomic DNA of the cultured cell or unicellular organism carrying the ccc DNA.
Standard protocols designed to simplify this process as far as possible have the problem that after the degradation step for genomic DNA the sample still comprises the degrading protein or fragments thereof. A partial answer to this problem is provided by such suppliers as Epicenter Technologies (Madison, Wisconsin, USA), which distributes an ATP-dependent DNAse under the tradename Plasmid-Safe.TM. for the separation of plasmid DNA (ccc DNA) from genomic DNA. The enzyme specifically cleaves linear DNA (such as genomic DNA) while leaving ccc DNA intact. The earliest identification of an enzyme of this class, namely of the ATP-dependent exonuclease RecBCD, was published in 1972 (Goldmark and Linn, J. Biol. Chem., 247, 1849-1860).
In spite of such advances, proteinaceous impurities contained in the ccc DNA sample solutions generate problems when the ccc DNA is used for subcloning purposes. In addition, such impurities may cause adverse side effects when used in in vivo applications.
Thus, the technical problem underlying the present invention was to improve the existing protocols for separating ccc DNA from genomic DNA. In accordance with the present invention it was found that a novel process comprising a sequence of steps discussed in more detail below not only provides ccc DNA of a high purity grade but also removes proteinaceous impurities that could interfere with subsequent cloning steps or in vivo applications.